The long-term research objects are to understand the cellular mechanism for synaptic co-transmission, its modulation, and their roles for the functions of nervous system. The research will determine the mechanistic details whereby presynaptic Ca2+ and its modulation differentially control the co-release of acetylcholine (ACh) and Luteinizing Hormone Release Hormone (LHRH) for bullfrog sympathetic preganglionic nerve terminals. Methods developed by the PI for selectively filling these cotransmitting terminals with DM-nitrophen, diazo-4 and fura-2 in their membrane impermeant forms will be used to manipulate uniformly or monitor directly the intraterminal Ca2+ transients while the release of ACh and LHRH from these terminals will be monitored as a fast nicotinic potential and a late slow LHRH potential/current respectively from ganglionic C neurons. The intraterminal Ca2+ transients will be evoked by electrical stimulation to the preganglionic nerve. By utilizing combinations of these techniques, the PI proposes to: I) Determine the differential Ca2+ requirement for release of ACh and LHRH from the same presynaptic terminals. The intraterminal Ca2+ transients will be manipulated uniformly by photolysis of DM-nitrophen while the release of ACh and LHRH from these terminals being monitored postsynaptically as either ACh- or LHRH-evoked potentials respectively. II) Examine the number of CA2+ binding sites required for LHRH release and the time course of LHRH release. The intraterminal Ca2+ transients will be either measured by fura-2 fluorimetry or terminated by diazo-4 at various time intervals after the end of the stimulation while release of LHRH being recorded postsynaptically. III) Systematically study the modulation of nerve evoked Ca2+ transient by endogenous neuropeptides and various Ca2+ channel blockers and its effects on LHRH release. Fura-2 fluorimetry will be used to dissect the effects of peptides including LHRH, neuropeptide Y (NPY), substance P and calcitonin gene-related peptide, and blockers to various voltage-gated Ca2+ channel on intraterminal Ca2+ transients. IV) Investigate the Ca2+ induced Ca2+ release in presynaptic terminals and its effect on LHRH release. Nerve stimulation, fura-2 fluorimetry and various pharmacological agents will be used to elucidate the capacity and kinetics of different intraterminal Ca2+-induced Ca2+ release processes. Specifically, since the tension of arteries in mammals are regulated by sympathetic neurons via the co-release of norepinephrine, NPY and possibly ATP, this project will generate insights into the neurogenic control of the vasculature. In general, many central and peripheral neurons contain and possibly release multiple neurotransmitters, thus information from these studies will lead to understanding of the mechanisms regulating cotransmission, which may be targets for malfunction in disease.